Essential bacterial genes and their use

ABSTRACT

Disclosed are two genes, termed “yphC” and “yqjK,” found in  Streptococcus pneumoniae , which are essential for survival for a wide range of bacteria. These genes and the essential polypeptides that they encode, as well as homologs and orthologs thereof, can be used to identify antibacterial agents for treating a broad spectrum of bacterial infections.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority under 35 U.S.C. §119(e) fromU.S. Ser. No 60/099,578, filed Sep. 9, 1998, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to essential bacterial genes and their usein identifying antibacterial agents.

BACKGROUND OF THE INVENTION

[0003] Bacterial infections may be cutaneous, subcutaneous, or systemic.Opportunistic bacterial infections proliferate, especially in patientsafflicted with AIDS or other diseases that compromise the immune system.Most bacteria that are pathogenic to humans are gram positive bacteria.The bacterium Streptococcus pneumoniae, for example, typically infectsthe respiratory tract and can cause lobar pneumonia, as well asmeningitis, sinusitis, and other infections.

SUMMARY OF THE INVENTION

[0004] The invention is based on the discovery of two genes in the grampositive bacterium Streptococcus pneumoniae that are essential for thesurvival of this and other bacteria. For convenience, these genes, yphCand yqjK, are collectively referred to herein as “essential” genes andthe polypeptides that these genes encode are referred to as “essential”polypeptides since Streptococcus pneumoniae cells lacking functionalyphC or yqjK genes are unable to survive.

[0005] The yphC and yqjK genes are useful molecular tools foridentifying similar genes in pathogenic microorganisms. The essentialpolypeptides that these genes encode are useful targets for identifyingcompounds that are inhibitors of the pathogens in which the essentialpolypeptides are expressed. Such compounds diminish bacterial growth byinhibiting the activity of an essential protein, or by inhibitingtranscription of an essential gene or translation of the mRNAtranscribed from the essential gene.

[0006] The invention, therefore, features an isolated yphC polypeptidehaving the amino acid sequence set forth in SEQ ID NO:2, as depicted inFIG. 1, or conservative variations thereof. An isolated nucleic acidencoding yphC also is included within the invention. In addition, theinvention includes (a) an isolated nucleic acid having the sequence ofSEQ ID NO:1, as depicted in FIG. 1, or degenerate variants thereof; (b)an isolated nucleic acid having the sequence of SEQ ID NO:1, ordegenerate variants thereof, wherein T is replaced by U; (c) nucleicacids complementary to (a) and (b); and (d) fragments of (a), (b), and(c) that are at least 15 base pairs in length and that hybridize understringent conditions, as described below, to genomic DNA encoding thepolypeptide of SEQ ID NO:2. The yphC polypeptide depicted in FIG. 1 is apartial sequence of the full-length polypeptide, which is depicted inFIGS. 2A-2B. The invention also features an isolated yphC polypeptidehaving the amino acid sequence set forth in SEQ ID NO:5, as depicted inFIGS. 2A-2B, or conservative variations thereof. An isolated nucleicacid encoding full-length yphC also is included within the invention. Inaddition, the invention includes (a) an isolated nucleic acid having thesequence of SEQ ID NO:4, as depicted in FIGS. 2A-2B, or degeneratevariants thereof; (b) an isolated nucleic acid having the sequence ofSEQ ID NO:4, or degenerate variants thereof, wherein T is replace by U;and (c) nucleic acids complementary to (a) and (b).

[0007] As described above for yphC, the invention includes an isolatednucleic acid encoding yqjK. In addition, the invention includes (a) anisolated nucleic acid having the sequence of SEQ ID NO:7, as depicted inFIG. 3, or degenerate variants thereof; (b) an isolated nucleic acidhaving the sequence of SEQ ID NO:7, or degenerate variants thereof,wherein T is replaced by U; (c) nucleic acids complementary to (a) and(b); and (d) fragments of (a), (b), and (c) that are at least 15 basepairs in length and that hybridize under stringent conditions, asdescribed below, to genomic DNA encoding the polypeptide of SEQ ID NO:8.These sequences are summarized in Table 1. TABLE 1 Essential NucleicAcids and Polypeptides SEQ ID SEQ ID NO. OF NO. OF AMINO SEQ ID NO. NON-Essential Nucleic FIG. ACID OF CODING CODING Acid or Polypeptide NO.SEQUENCE STRAND STRAND yphC-partial 1 2 1 3 sequence yphC-full-length2A-2B 5 4 6 yqjK 3 8 7 9

[0008] Identification of these essential genes allows homologs of theessential genes to be found in other strains within the species, and itallows orthologs of the essential genes to be found in other organisms(e.g., Bacillus sp., H. influenzae, H. pylori, and E. coli). While“homologs” are structurally similar genes contained within theStreptococcus species, “orthologs” are functionally equivalent genesfrom other species, as determined, for example, in a standardcomplementation assay. Thus, the essential polypeptides can be used notonly as a model for identifying similar genes in other Streptococcusstrains, but also to identify homologs and orthologs of essential genesin other species (e.g., other gram positive bacteria, particularly thosebacteria that are pathogenic to humans, and other bacteria generally).Such orthologs can be identified, for example, in a conventionalcomplementation assay. In addition, or alternatively, such orthologs canbe expected to exist in bacteria in the same branch of the phylogenetictree, as set forth, for example, atftp://ftp.cme.msu.edu/pub/RDP/SSU_rRNA/SSU/Prok.phylo. For example, B.subtilis is in the B. subtilis subgroup of the B. subtilis group in theBacillus-Lactobaccillus-Streptococcus Subdivision of the Gram positivephylum. Likewise, S. pneumoniae belong to the Stc. pneumonia subgroup ofStreptococci, which also are in the Bacillus-Lactobacillus-Streptococcussubdivision of the Gram positive phylum. E. coli belong to theEscherichia Salmonella group of the Enterics and relatives within theGamma subdivision of the Purple bacteria. Other bacteria within the samephylum (particularly, bacteria within the same subdivision, group, orsubgroup) can be expected to contain an ortholog of the yphC and/or yqjKgenes described herein.

[0009] Examples of orthologs of the Streptococcus yphC and yqjK genesare summarized in Table 2. As shown in Table 2, the Streptococcus geneyphC has an ortholog in B. subtilis, termed “B-yphC,” and an ortholog inE. coli, termed “yfgK,” which is also known as “f503.” The Streptococcusgene yqjK also has an ortholog in B. subtilis, termed “B-yqjK,” and anortholog in E. coli, termed “elaC,” which is also known as “o311.” Asdiscussed below, orthologs of essential genes may themselves beessential or non-essential in the organism in which they are found.

[0010] As determined by the experiments described below, the B-yphC,yfgK, and B-yqjK orthologs are essential for survival of the bacteria inwhich they are found. Thus, these essential orthologous genes and thepolypeptides encoded by these orthologs can be used to identifycompounds that inhibit the growth of the host organism (e.g., compoundsthat inhibit the activity of an essential protein, or inhibittranscription of an essential gene). TABLE 2 Orthologs of yphC and yqjKSEQ ID SEQ ID SEQ ID NO. of NO. of NO. of Amino Nucleic Non- NucleicAcid Acid Coding Acid or FIG. Sequence Sequence Strand Poly- Number ofof of of peptide Ortholog Ortholog Ortholog Ortholog Ortholog yphC B.subtilis 4A-4B 11 10 12 B-yphC GenBank Accession No. Z99115 yphC E. coli5A-5B 14 13 15 yfgK GenBank Accession No. AE000337 yqjK B. subtilis 6 1716 18 B-yqjK GenBank Accession No. Z99116 yqjK E. coli 7 20 19 21 elaCGenBank Accession No. AE000316

[0011] The yphC polypeptides and genes described herein include thepolypeptides and genes set forth in FIGS. 1 and 2A-2B herein, as well asisozymes, variants, and conservative variations of the sequences setforth in FIGS. 1 and 2A-2B. The invention includes various isozymes ofyphC and yqjK. For example, the invention includes a gene that encodesan essential polypeptide but which gene includes one or more pointmutations, deletions, or promoter variants, provided that the resultingessential polypeptide retains a biological function of an essentialpolypeptide.

[0012] The yphC polypeptide has structural characteristics of knownGTPases. Using BLAST analysis, the yphC polypeptide has been shown tocontain two domains that are predicted to be GTPase domains, and yphCdisplays GTPase activity in vitro. This GTPase activity is linked to theessentiality of the yphC polypeptide. When point mutations are made ineach GTPase domain of yphC such that the mutants are unable to bind GTP,such mutants no longer are able to complement a bacterial strain thatlacks yphC. The yqjK polypeptide has structural characteristics of knownsulfatases. Thus, the various isozymes, variants, and conservativevariations of the yphC and yqjK sequences set forth in FIGS. 1 and 2A-2Bretain a biological function of yphC or yqjK as determined, for example,in an assay of GTPase or sulfatase activity, or in a conventionalcomplementation assay. Suitable GTPase and sulfatase activity assays arewell known in the art (see, e.g., Bollag, et al., Meth. Enzymol. 255:161(1995) and Barbeyron, et al., Microbiol. 141:2897 (1995), incorporatedherein by reference). The GTPase activity of yphC can also be assayedusing a conventional Malachite Green phosphorelease assay (see, e.g.,Lanzetta et al., 1979, Analytical Biochemistry 100:95-97). The inclusionof KCl in such an assay leads to an approximately 70-fold stimulation ofGTPase activity, and thus provides a sensitive assay for detection ofGTP activity.

[0013] Also encompassed by the term yphC gene are degenerate variants ofthe nucleic acid sequences set forth in FIGS. 1 and 2A-2B (SEQ ID NO:1and 4). Degenerate variants of a nucleic acid sequence exist because ofthe degeneracy of the amino acid code; thus, those sequences that varyfrom the sequence represented by SEQ ID NO:1 and 4, but whichnonetheless encode a yphC polypeptide are included within the invention.

[0014] Likewise, because of the similarity in the structures of aminoacids, conservative variations (as described herein) can be made in theamino acid sequence of the yphC polypeptide while retaining the functionof the polypeptide (e.g., as determined in a conventionalcomplementation assay). Other yphC polypeptides and genes identified inadditional bacterial strains may be such conservative variations ordegenerate variants of the particular yphC polypeptide and nucleic acidset forth in FIGS. 1 and 2A-2B (SEQ ID NOs:1-6). The yphC polypeptideand gene share at least 80%, e.g., 90%, sequence identity with SEQ IDNOs:2 and 1, respectively, or SEQ ID NOs: 5 and 4,-respectively.Regardless of the percent sequence identity between the yphC sequenceand the sequences represented by SEQ ID NOs:1, 2, 4, and 5, the yphCgenes and polypeptides encompassed by the invention preferably are ableto complement for the lack of yphC function (e.g., in atemperature-sensitive mutant) in a standard complementation assay.

[0015] Additional yphC genes that are identified and cloned fromadditional bacterial strains, and pathogenic, gram-positive strains inparticular, can be used to produce yphC polypeptides for use in thevarious methods described herein, e.g., for identifying antibacterialagents. Likewise, the term yqjK encompasses isozymes, variants, andconservative variations of the sequences depicted in FIG. 3.

[0016] In various embodiments, the essential polypeptide used in theassays described herein is derived from a non-pathogenic or pathogenicgram positive bacterium. For example, the polypeptide can be derivedfrom a Streptococcus strain, such as Streptococcus pneumoniae,Streptococcus pyogenes, Streptococcus agalactiae, Streptococcusendocarditis, Streptococcus faecium, Streptococcus sangus, Streptococcusviridans, and Streptococcus hemolyticus. Orthologs of the yphC and yqjKgenes can be derived from a wide spectrum of bacteria, such as E. coliand Bacillus subtilis.

[0017] Having identified the yphC and yqjK genes described herein asbeing essential for survival, these essential genes and the polypeptidesencoded by these essential genes and their essential homologs andorthologs can be used to identify antibacterial agents. Suchantibacterial agents can readily be identified with high throughputassays to detect inhibition of the metabolic pathway in which theessential polypeptide participates. This inhibition can be caused bysmall-molecules interacting with (e.g., binding directly or indirectlyto) the essential polypeptide or other essential polypeptides in thatpathway.

[0018] An exemplary method for identifying antibacterial compoundsinvolves screening for small molecules that specifically interact with(i.e., bind directly or indirectly to) the essential polypeptide. Avariety of suitable interaction and binding assays are known in the artas described, for example, in U.S. Pat. Nos. 5,585,277 and 5,679,582,incorporated herein by reference. For example, in various conventionalassays, test compounds can be assayed for their ability to interact withan essential polypeptide by measuring the ability of the small moleculeto stabilize the essential polypeptide in its folded, rather thanunfolded, state. More specifically, the degree of protection fromunfolding that is afforded by the test compound can be measured. Testcompounds that bind the essential polypeptide with high affinity cause,for example, a large shift in the temperature at which the polypeptideis denatured. Test compounds that stabilize the essential polypeptide ina folded state can be further tested for antibacterial activity in astandard susceptibility assay.

[0019] Another exemplary method for identifying antibacterial agentsinvolves measuring the ability of a test compound to bind to one of theessential polypeptides described herein. Binding can be assayed in aconventional capillary electrophoresis assay in which binding of thetest compound to the essential polypeptide changes the electrophoreticmobility of the essential polypeptide.

[0020] Another suitable method for identifying inhibitors of theessential polypeptides involves identifying a biochemical activity ofthe essential polypeptide and then screening for small moleculeinhibitors of the activity using; for example, a high throughputscreening method. The yphC polypeptide has structural characteristics ofknown GTPases and displays GTPase activity in vitro. Therefore,inhibitors of this polypeptide therefore can be identified by theirability to inhibit the GTPase activity of yphC in a conventional assayof GTPase activity. Suitable assays have been described (e.g., Gollag etal., Meth. Enzymol. 255: 161-170, 1995, which is incorporated herein byreference). A detailed example of a suitable assay is set forth below.

[0021] The yqjK polypeptide has structural characteristics of sulfatasesand is expected to function as a sulfatase. Accordingly, inhibitors ofthe yqjK polypeptide can be identified by assaying for the ability ofthe test compound to inhibit the sulfatase activity of yqjK. An exampleof a suitable assay is described by Barbeyron et al., Microbiol.141:2897-2904, 1995, which is incorporated herein by reference.

[0022] The invention also includes a method for identifying anantibacterial agent which method entails: (a) contacting an essentialpolypeptide, or homolog or orthologs thereof, with a test compound; (b)detecting binding of the test compound to the polypeptide or homolog orortholog; and, optionally, (c) determining whether a test compound thatbinds to the polypeptide or homolog or ortholog inhibits growth ofbacteria, relative to growth of bacteria cultured in the absence of thetest compound that binds to the polypeptide or homolog or ortholog, asan indication that the test compound is an antibacterial agent.

[0023] In another suitable assay, a promoter that responds to depletionof the essential polypeptide by upregulation or downregulation is linkedto a reporter gene. To identify a promoter that is up- or down-regulatedby the depletion of an essential protein, the gene encoding theessential protein is deleted from the genome and replaced with a versionof the gene in which the sequence encoding the essential protein isoperably linked to a regulatable promoter. The cells containing thisregulatable genetic construct are kept alive by the essentialpolypeptide produced from the genetic construct containing theregulatable promoter. However, the regulatable promoter allows theexpression of the essential polypeptide to be reduced to a level thatcauses growth inhibition. Total RNA prepared from bacteria under suchgrowth-limiting conditions is compared with RNA from wild-type cells.Standard methods of transcriptional profiling can be used to identifymRNA species that are either more or less abundant (i.e., up- ordown-regulated) when expressed under the limiting conditions. Genomicsequence information, e.g., from GenBank, can be used to identify thepromoter that drives expression of the identified RNA species. Suchpromoters are up- or down-regulated by depletion of the essentialpolypeptide.

[0024] Having identified a promoter(s) that is up- or down-regulated bydepletion of the essential polypeptide, the promoter(s) is operablylinked to a reporter gene (e.g., β-galactosidase, gus, or greenfluorescent protein (GFP)). A bacterial strain containing this reportergene construct is then exposed to test compounds. Compounds that inhibitthe essential polypeptide (or other polypeptides in the essentialpathway in which the essential polypeptide participates) cause afunctional depletion of the essential polypeptide and therefore lead toan upregulation or downregulation of expression the reporter gene.Compounds that inhibit the essential polypeptides in such an assay areexpected to be antibacterial and can be further tested, if desired, instandard susceptibility assays.

[0025] In a related method for identifying antibacterial compounds, theessential polypeptides are used to isolate peptide or nucleic acidligands that specifically bind the essential polypeptides. These peptideor nucleic acid ligands are then used in a displacement screen toidentify small molecules that interact with the essential polypeptide.Such assays can be carried out essentially as described above.

[0026] In still another method, interaction of a test compound with anessential polypeptide (i.e., direct or indirect binding) can be detectedin a conventional two-hybrid system for detecting protein/proteininteractions (e.g., in yeast or mammalian cells). A test compound foundto interact with the essential polypeptide can be further tested forantibacterial activity in a conventional susceptibility assay.Generally, in such two-hybrid methods, (a) the essential polypeptide isprovided as a fusion protein that includes the polypeptide fused to (i)a transcription activation domain of a transcription factor or (ii) aDNA-binding domain of a transcription factor; (b) the test polypeptideis provided as a fusion protein that includes the test polypeptide fusedto (i) a transcription activation domain of a transcription factor or(ii) a DNA-binding domain of a transcription factor; and (c) binding ofthe test polypeptide to the polypeptide is detected as a reconstitutionof a transcription factor. Homologs and orthologs of the essentialpolypeptides can be used in similar methods. Reconstitution of thetranscription factor can be detected, for example, by detectingtranscription of a gene that is operably linked to a DNA sequence boundby the DNA-binding domain of the reconstituted transcription factor(See, for example, White, 1996, Proc. Natl. Acad. Sci. 93:10001-10003and references cited therein and Vidal et al., 1996, Proc. Natl. Acad.Sci. 93:10315-10320).

[0027] In an alternative method, an isolated nucleic acid moleculeencoding an essential polypeptide is used to identify a compound thatdecreases the expression of an essential polypeptide in vivo. Suchcompounds can be used as antibacterial agents. To identify suchcompounds, cells that express an essential polypeptide are cultured,exposed to a test compound (or a mixture of test compounds), and thelevel of expression or activity is compared with the level of essentialpolypeptide expression or activity in cells that are otherwise identicalbut that have not been exposed to the test compound(s). Many standardquantitative assays of gene expression can be utilized in this aspect ofthe invention.

[0028] To identify compounds that modulate expression of an essentialpolypeptide (or homologous or orthologous sequence), the testcompound(s) can be added at varying concentrations to the culture mediumof cells that express an essential polypeptide (or homolog or ortholog),as described herein. Such test compounds can include small molecules(typically, non-protein, non-polysaccharide chemical entities),polypeptides, and nucleic acids. The expression of the essentialpolypeptide is then measured, for example, by Northern blot PCR analysisor RNAse protection analyses using a nucleic acid molecule of theinvention as a probe. The level of expression in the presence of thetest molecule, compared with the level of expression in its absence,will indicate whether or not the test molecule alters the expression ofthe essential polypeptide. Because the yphC and yqjK polypeptides areessential for survival, test compounds that inhibit the expressionand/or function of the essential polypeptide, or of an essential homologor ortholog thereof, will inhibit growth of, or kill, the cells thatexpress such polypeptides.

[0029] The polypeptides encoded by essential genes also can be used,separately or together, in assays to identify test compounds thatinteract with these polypeptides. Test compounds that interact withthese polypeptides then can readily be tested, in conventional assays,for their ability to inhibit bacterial growth. Test compounds thatinteract with the essential polypeptides are candidate antibacterialagents, in contrast to compounds that do not interact with the essentialpolypeptides. As described herein, any of a variety of art-known methodscan be used to assay for the interaction of test compounds with theessential polypeptides.

[0030] Typically, the test compound will be a small organic molecule.Alternatively, the test compound can be a test polypeptide (e.g., apolypeptide having a random or predetermined amino acid sequence; or anaturally-occurring or synthetic polypeptide) or a nucleic acid, such asa DNA or RNA molecule. The test compound can be a naturally-occurringcompound or it can be synthetically produced, if desired. Syntheticlibraries, chemical libraries, and the like can be screened to identifycompounds that bind the essential polypeptide. More generally, bindingof test a compound to the polypeptide, homolog, or ortholog can bedetected either in vitro or in vivo. If desired, the above-describedmethods for identifying compounds that modulate the expression of thepolypeptides of the invention can be combined with measuring the levelsof the essential polypeptides expressed in the cells, e.g., byperforming a Western blot analysis using antibodies that bind anessential polypeptide.

[0031] Regardless of the source of the test compound, the essentialpolypeptides described herein can be used to identify compounds thatinhibit the activity of an essential protein or transcription of anessential gene or translation of the mRNA transcribed from the essentialgene. These antibacterial agents can be used to inhibit a wide spectrumof pathogenic or non-pathogenic bacterial strains.

[0032] In other embodiments, the invention includes pharmaceuticalformulations that include a pharmaceutically acceptable excipient and anantibacterial agent identified using the methods described herein. Inparticular, the invention includes pharmaceutical formulations thatcontain antibacterial agents that inhibit the growth of, or kill,pathogenic bacterial strains (e.g., pathogenic gram positive bacterialstrains such as pathogenic Streptococcus strains). Such pharmaceuticalformulations can be used in a method of treating a bacterial infectionin an organism (e.g., a Streptococcus infection). Such a method entailsadministering to the organism a therapeutically effective amount of thepharmaceutical formulation, i.e., an amount sufficient to amelioratesigns and/or symptoms of the bacterial infection. In particular, suchpharmaceutical formulations can be used to treat bacterial infections inmammals such as humans and domesticated mammals (e.g., cows, pigs, dogs,and cats), and in plants. The efficacy of such antibacterial agents inhumans can be estimated in an animal model system well known to those ofskill in the art (e.g., mouse and rabbit model systems of, for example,streptococcal pneumonia).

[0033] Various affinity reagents that are permeable to the microbialmembrane (i.e., antibodies and antibody fragments) are useful inpracticing the methods of the invention. For example polyclonal andmonoclonal antibodies that specifically bind to the yphC polypeptide oryqjK polyglypeptide can facilitate detection of essential polypeptidesin various bacterial strains (or extracts thereof). These antibodiesalso are useful for detecting binding of a test compound to essentialpolypeptides (e.g., using the assays described herein). In addition,monoclonal antibodies that bind essential polypeptides can themselves beused as antibacterial agents.

[0034] The invention further features methods of identifying from alarge group of mutants those strains that have conditional lethalmutations. In general, the gene and corresponding gene product aresubsequently identified, although the strains themselves can be used inscreening or diagnostic assays. The mechanism(s) of action for theidentified genes and gene products provide a rational basis for thedesign of antibacterial therapeutic agents. These antibacterial agentsreduce the action of the gene product in a wild type strain, andtherefore are useful in treating a subject with that type, or asimilarly susceptible type, of infection by administering the agent tothe subject in a pharmaceutically effective amount. Reduction in theaction of the gene product includes competitive inhibition of the geneproduct for the active site of an enzyme or receptor; non-competitiveinhibition; disrupting an intracellular cascade path which requires thegene product; binding to the gene product itself, before or afterpost-translational processing; and acting as a gene product mimetic,thereby down-regulating the activity. Therapeutic agents includemonoclonal antibodies raised against the gene product.

[0035] Furthermore, the presence of the gene sequence in certain cells(e.g., a pathogenic bacterium of the same genus or similar species), andthe absence or divergence of the sequence in host cells can bedetermined, if desired. Therapeutic agents directed toward genes or geneproducts that are not present in the host have several advantages,including fewer side effects, and a lower overall required dosage.

[0036] Nucleic acids include both RNA and DNA, including genomic DNA andsynthetic (e.g., chemically synthesized) DNA. Nucleic acids can bedouble-stranded or single-stranded. Where single-stranded, the nucleicacid may be a sense strand or an antisense strand. Nucleic acids can besynthesized using oligonucleotide analogs or derivatives (e.g., inosineor phosphorothioate nucleotides). Such oligonucleotides can be used, forexample, to prepare nucleic acids that have altered base-pairingabilities or increased resistance to nucleases.

[0037] An isolated nucleic acid is a DNA or RNA that is not immediatelycontiguous with both of the coding sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived.Thus, in one embodiment, an isolated nucleic acid includes some or allof the 5′ non-coding (e.g., promoter) sequences that are immediatelycontiguous to the coding sequence. The term therefore includes, forexample, a recombinant DNA that is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other sequences. It also includes arecombinant DNA that is part of a hybrid gene encoding an additionalpolypeptide sequence. The term “isolated” can refer to a nucleic acid orpolypeptide that is substantially free of cellular material, viralmaterial, or culture medium (when produced by recombinant DNAtechniques), or chemical precursors or other chemicals (when chemicallysynthesized). Moreover, an isolated nucleic acid fragment is a nucleicacid fragment that is not naturally occurring as a fragment and wouldnot be found in the natural state.

[0038] A nucleic acid sequence that is substantially identical to anessential nucleotide sequence is at least 80% (e.g., at least 85%)identical to the nucleotide sequence of yphC or yqjK as represented bythe SEQ ID NOs listed in Table 1, as depicted in FIGS. 1-3. For purposesof comparison of nucleic acids, the length of the reference nucleic acidsequence will generally be at least 40 nucleotides, e.g., at least 60nucleotides or more nucleotides.

[0039] To determine the percent identity of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of overlapping positions×100). Preferably,the two sequences are the same length.

[0040] The determination of percent identity or homology between twosequences can be accomplished using a mathematical algorithm. Asuitable, mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Nat'lAcad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to yphC or yqjK nucleic acid moleculesof the invention. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to yphC or yqjK protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used.

[0041] The percent identity between two sequences can be determinedusing the techniques described above, with or without allowing gaps. Incalculating percent identity, only exact matches are counted.

[0042] The essential polypeptides useful in practicing the inventioninclude, but are not limited to, recombinant polypeptides and naturalpolypeptides. Also useful in the invention are nucleic acid sequencesthat encode forms of essential polypeptides in which naturally occurringamino acid sequences are altered or deleted. Preferred nucleic acidsencode polypeptides that are soluble under normal physiologicalconditions. Also within the invention are nucleic acids encoding fusionproteins in which a portion of an essential polypeptide is fused to anunrelated polypeptide (e.g., a marker polypeptide or a fusion partner)to create a fusion protein. For example, the polypeptide can be fused toa hexa-histidine tag to facilitate purification of bacterially expressedpolypeptides, or to a hemagglutinin tag to facilitate purification ofpolypeptides expressed in eukaryotic cells. The invention also includes,for example, isolated polypeptides (and the nucleic acids that encodethese polypeptides) that include a first portion and a second portion;the first portion includes an essential polypeptide, and the secondportion includes an immunoglobulin constant (Fc) region or a detectablemarker.

[0043] The fusion partner can be, for example, a polypeptide whichfacilitates secretion, e.g., a secretory sequence. Such a fusedpolypeptide is typically referred to as a preprotein. The secretorysequence can be cleaved by the host cell to form the mature protein.Also within the invention are nucleic acids that encode an essentialpolypeptide fused to a polypeptide sequence to produce an inactivepreprotein. Preproteins can be converted into the active form of theprotein by removal of the inactivating sequence.

[0044] The invention also includes nucleic acids that hybridize, e.g.,under stringent hybridization conditions (as defined herein) to all or aportion of the nucleotide sequences represented by SEQ ID NO:1 or 7, ortheir complements. The hybridizing portion of the hybridizing nucleicacids is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides inlength. The hybridizing portion of the hybridizing nucleic acid is atleast 80%, e.g., at least 95%, or at least 98%, identical to thesequence of a portion or all of a nucleic acid encoding an essentialpolypeptide or its complement. Hybridizing nucleic acids of the typedescribed herein can be used, for example, as a cloning probe, a primer(e.g., a PCR primer), or a diagnostic probe. Nucleic acids thathybridize to the nucleotide sequences represented by SEQ ID NOs: 1 and 7are considered “antisense oligonucleotides.”

[0045] Also part of in the invention are various engineered cells, e.g.,transformed host cells, that contain an essential nucleic acid describedherein. A transformed cell is a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, anucleic acid encoding an essential polypeptide. Both prokaryotic andeukaryotic cells are included, e.g., bacteria, such as Streptococcus,Bacillus, and the like.

[0046] Also within the invention are genetic constructs (e.g., vectorsand plasmids) that include a nucleic acid of the invention that isoperably linked to a transcription and/or translation sequence to enableexpression, e.g., expression vectors. A selected nucleic acid, e.g., aDNA molecule encoding an essential polypeptide, is “operably linked” toa transcription and/or translation sequence when it is positionedadjacent to one or more sequence elements, e.g., a promoter, whichdirect transcription and/or translation of the sequence such that thesequence elements can control transcription and/or translation of theselected nucleic acid.

[0047] The invention also features purified or isolated polypeptidesencoded by the essential genes yphC and yqjK. The terms “protein” and“polypeptide” both refer to any chain of amino acids, regardless oflength or post-translational modification (e.g., glycosylation orphosphorylation). Thus, the terms yphC polypeptide and yqjK polypeptideinclude full-length, naturally occurring, isolated yphC and yqjKproteins, respectively, as well as recombinantly or syntheticallyproduced polypeptides that correspond to the full-length, naturallyoccurring proteins, or to a portion of the naturally occurring orsynthetic polypeptide (provided that a portion of the yphC polypeptideincludes a portion of the sequence depicted in FIG. 1).

[0048] A purified or isolated compound is a composition that is at least60% by weight the compound of interest, e.g., an essential polypeptideor antibody. Preferably the preparation is at least 75% (e.g., at least90%, 95%, or even 99%) by weight the compound of interest. Purity can bemeasured by any appropriate standard method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0049] Preferred essential polypeptides include a sequence substantiallyidentical to all or a portion of a naturally occurring essentialpolypeptide, e.g., including all or a portion of the sequences shown inFIGS. 1, 2A-2B, and 3 (provided that a portion of the yphC polypeptideincludes a portion of the sequence depicted in FIG. 1). Polypeptides“substantially identical” to the essential polypeptide sequencesdescribed herein have an amino acid sequence that is at least 80%identical to the amino acid sequence of the essential polypeptidesrepresented by the SEQ ID NOs listed in Table 1 (measured as describedherein). The new polypeptides can also have a greater percentageidentity, e.g., 85%, 90%, 95%, or even higher. For purposes ofcomparison, the length of the reference essential polypeptide sequencewill generally be at least 16 amino acids, e.g., at least 20 or 25 aminoacids.

[0050] In the case of polypeptide sequences that are less than 100%identical to a reference sequence, the non-identical positions arepreferably, but not necessarily, conservative substitutions for thereference sequence. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine.

[0051] Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference polypeptide. Thus, a polypeptidethat is 50% identical to a reference polypeptide that is 100 amino acidslong can be a 50 amino acid polypeptide that is completely identical toa 50 amino acid long portion of the reference polypeptide.Alternatively, it can be a 100 amino acid long polypeptide that is 50%identical to the reference polypeptide over its entire length. Ofcourse, other polypeptides also will meet the same criteria.

[0052] The invention also features purified or isolated antibodies thatspecifically bind to an essential polypeptide. An antibody “specificallybinds” to a particular antigen, e.g., an essential polypeptide, when itbinds to that antigen, but does not substantially recognize and bind toother molecules in a sample, e.g., a biological sample, that naturallyincludes an essential polypeptide.

[0053] In another aspect, the invention features a method for detectingan essential polypeptide in a sample. This method includes: obtaining asample suspected of containing an essential polypeptide; contacting thesample with an antibody that specifically binds to an essentialpolypeptide under conditions that allow the formation of complexes of anantibody and the essential polypeptide; and detecting the complexes, ifany, as an indication of the presence of an essential polypeptide in thesample.

[0054] Also encompassed by the invention is a method of obtaining a generelated to an essential gene. Such a method entails obtaining a labeledprobe that includes an isolated nucleic acid which encodes all or aportion of an essential nucleic acid, or a homolog thereof; screening anucleic acid fragment library with the labeled probe under conditionsthat allow hybridization of the probe to nucleic acid fragments in thelibrary, thereby forming nucleic acid duplexes; isolating labeledduplexes, if any; and preparing a full-length gene sequence from thenucleic acid fragments in any labeled duplex to obtain a gene related tothe essential gene. Alternatively, such related genes can be identifiedby carrying out a BLAST search of various sequenced bacterial genomes,as described above.

[0055] The invention offers several advantages. For example, the methodsfor identifying antibacterial agents can be configured for highthroughput screening of numerous candidate antibacterial agents. Becausethe essential genes disclosed herein are thought to be highly conserved,antibacterial drugs targeted to these genes or their gene products areexpected to have antibacterial activity against a wide range ofbacteria.

[0056] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described herein. All publications, patent applications,patents, and other references mentioned herein are incorporated hereinby reference in their entirety. In the case of a conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative and are not intendedto limit the scope of the invention, which is defined by the claims.

[0057] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058]FIG. 1 is a representation of the amino acid and nucleic acidsequences of the yphC polypeptide and coding and non-coding strands ofthe yphC gene from a Streptococcus pneumoniae strain (SEQ ID NOs:2, 1,and 3, respectively).

[0059] FIGS. 2A-2B are a representation of the full-length amino acidand nucleic acid sequences of the yphC polypeptide and coding andnon-coding strands of the yphC gene from a Streptococcus pneumoniaestrain (SEQ ID NOs:5, 4, and 6, respectively).

[0060]FIG. 3 is a representation of the amino acid and nucleic acidsequences of the yqjK polypeptide and coding and non-coding strands ofthe yqjK gene from a Streptococcus pneumoniae strain (SEQ ID NOs:8, 7,and 9, respectively),

[0061] FIGS. 4A-4B are a representation of the amino acid and nucleicacid sequences of the B-yphC polypeptide and coding and non-codingstrands of the B-yphC gene from a B. subtilis strain (SEQ ID Nos:11, 10,and 12, respectively).

[0062] FIGS. 5A-5B are a representation of the amino acid and nucleicacid sequences of the yfgK polypeptide and coding and non-coding strandsof the yfgK gene from an E. coli strain (SEQ ID Nos:14, 13, and 15,respectively).

[0063]FIG. 6 is a representation of the amino acid and nucleic acidsequences of the B-yqjK polypeptide and coding and non-coding strands ofthe B-yqjk gene from a B. subtilis strain (SEQ ID Nos:17, 16, and 18,respectively).

[0064]FIG. 7 is a representation of the amino acid and nucleic acidsequences of the elaC polypeptide and gene from an E. coli strain (SEQID Nos:20, 19, and 21, respectively).

[0065]FIG. 8 is a schematic representation of the PCR strategy used toproduce DNA molecules used for targeted deletions of essential genes inStreptococcus pneumoniae.

[0066]FIG. 9 is a schematic representation of the strategy used toproduce targeted deletions of essential genes in Streptococcuspneumoniae.

[0067]FIG. 10 is a schematic representation of the strategy used toobtain non-polar gene deletions of yphC and yqjK in B. subtilis.

[0068] FIGS. 11A-11C are schematic representations of the strategy usedto construct conditional null mutants of the yphC and yqjK genes.

[0069]FIG. 12 is a schematic representation of the general strategy usedto obtain deletions of essential genes in E. coli and shows theessential phenotype of the E. coli yfgK gene, which is an ortholog ofthe S. pneumoniae yphC gene.

DETAILED DESCRIPTION OF THE INVENTION

[0070] At least two genes in the bacterium Streptococcus pneumoniae havebeen found to be essential for the survival of these bacteria. Theseso-called essential genes, yphC and yqjK, encode what are referred toherein as essential polypeptides. The yphC and yqjK genes are usefulmolecular tools for identifying similar genes in pathogenicmicroorganisms, such as pathogenic strains of Bacillus. The essentialpolypeptides are useful targets for identifying compounds that areinhibitors of the pathogens in which the essential polypeptides areexpressed.

[0071] Identifying Essential Streptococcus Genes

[0072] As shown by the experiments described below, both the yphC andyqjK genes are essential for survival of Streptococcus pneumoniae.Streptococcus pneumoniae is available from the ATCC. In general, and forthe examples set forth below, essential genes can be identified bycreating targeted deletions of genes of interest in a bacterium, e.g.,S. pneumoniae. These genes of interest were selected as follows. Usingstandard molecular biology techniques, a library containing fragments ofthe Streptococcus pneumoniae genome was made, using M13 phage or plasmidDNA as the vector. Open reading frames (ORFs) contained within thislibrary were randomly sequenced, using primers that hybridized to thevector. The genes of interest selected for targeted deletion satisfiedfour criteria, as determined by comparing the sequences with the GenBankdatabase of nucleotide sequences: (i) the ORF had no known function;(ii) the ORF had an ortholog in Bacillus subtilis; (iii) the ORF wasconserved in other bacteria, with p<10⁻¹⁰; and (iv) the ORF had noeukaryotic ortholog, with p>10⁻³. The Streptococcus genes yphC and yqjKmet each of these criteria, suggesting that a compound that inhibitedthe yphC or yqjK genes or gene products would have a broad spectrum ofantibacterial activity.

[0073] The yphC and yqjK genes each were replaced with a nucleic acidsequence conferring resistance to the antibiotic erythromycin (an “erm”gene). Other genetic markers can be used in lieu of this particularantibiotic resistance marker. Polymerase chain reaction (PCR)amplification was used to make a targeted deletion in the Streptococcusgenomic DNA, as shown in FIG. 8. Several PCR reactions were used toproduce the DNA molecules needed to carry out target deletion of thegenes of interest. First, using primers 5 and 6, an erm gene wasamplified from pIL252 from B. subtilis (available from the BacillusGenetic Stock Center, Columbus, Ohio). Primer 5 consists of 21nucleotides that are identical to the promoter region of the erm geneand complementary to Sequence A. Primer 5 has the sequence 5′GTG TTC GTGCTG ACT TGC ACC3′ (SEQ ID NO:22). Primer 6 consists of 21 nucleotidesthat are complementary to the 3′ end of the erm gene. Primer 6 has thesequence 5′GAA TTA TTT CCT CCC GTT AAA3′ (SEQ ID NO:23). PCRamplification of the erm gene was carried out under the followingconditions: 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, and72° C. for 1.5 minutes, followed by one cycle of 72° C. for 10 minutes.

[0074] In the second and third PCR reactions, sequences flanking thegene of interest were amplified and produced as hybrid DNA moleculesthat also contained a portion of the erm gene. The second reactionproduced a double-stranded DNA molecule (termed “Left FlankingMolecule”) that includes sequences upstream of the 5′ end of the gene ofinterest and the first 21 nucleotides of the erm gene. As shown in FIG.8, this reaction utilized primer 1, which is 21 nucleotides in lengthand identical to a sequence that is located approximately 500 bpupstream of the translation start site of the gene of interest. Primers1 and 2 are gene-specific and have the sequences 5′TGA AGC CTG TCA AGGACG AGG3′ (SEQ ID NO:24) and 5′CCT TAC GTG GTC GAA TTG TGG3′ (SEQ IDNO:25), respectively, for yqjK. For yphC, primers 1 and 2 have thesequences 5′TGT ATG AAT TGG TAC CTC AAG3′ (SEQ ID NO:26) and 5′ACA ATGGCA ATA GTT GGT AGG3′ (SEQ ID NO:27), respectively. Primer 2 is 42nucleotides in length, with 21 of the nucleotides at the 3′ end of theprimer being complementary to the 5′ end of the sense strand of the geneof interest. The 21 nucleotides at the 5′ end of the primer wereidentical to Sequence A and are therefore complementary to the 5′ end ofthe erm gene. Thus, PCR amplification using primers 1 and 2 produced theleft flanking DNA molecule, which is a hybrid DNA molecule containing asequence located upstream of the gene of interest and 21 base pairs ofthe erm gene, as shown in FIG. 8.

[0075] The third PCR reaction was similar to the second reaction, butproduced the right flanking DNA molecule, shown in FIG. 8. The rightflanking DNA molecule contains 21 base pairs of the 3′ end of the ermgene, a 21 base pair portion of the 3′ end of the gene of interest, andsequences downstream of the gene of interest. This right flanking DNAmolecule was produced with gene-specific primers 3 and 4. For yqjK,primers 3 and 4 have the sequences 5′GTG GAA ATC TAG CAG TCA CAG3′ (SEQID NO:28) and 5′ATC TGG TTC TAG CAG GAA GCG3′ (SEQ ID NO:29),respectively. For yphC, primers 3 and 4 have the sequences 5′CAT TGC CAGTCC TGT TGC TGG3′ (SEQ ID NO:30) and 5′ ATG GCA TCC ATG ACA TCG3′ (SEQID NO:31), respectively. Primer 3 is 42 nucleotides; the 21 nucleotidesat the 5′ end of primer 3 are identical to Sequence B and therefore areidentical to the 3′ end of the erm gene. The 21 nucleotides at the 3′end of primer 3 are identical to the 3′ end of the gene of interest.Primer 4 is 21 nucleotides in length and is complementary to a sequencelocated approximately 500 bp downstream of the gene of interest.

[0076] PCR amplification of the left and right flanking DNA moleculeswas carried out, separately, in 50 μl reaction mixtures containing: 1 μlStreptococcus pneumoniae (RX1) DNA (0.25 μg), 2.5 μl primer 1 or primer4 (10 pmol/μl), 2.5 μl primer 2 or primer 3 (20 pmol/μl), 1.2 μl amixture dNTPs (10 mM each), 37 μl H₂O, 0.7 μl Taq polymerase (5U/μl),and 5 μl 10× Taq polymerase buffer (10 mM Tris, 50 mM KCl, 2.5 mMMgCl₂). The left and right flanking DNA molecules were amplified usingthe following PCR cycling program: 95° C. for 2 minutes; 72° C. for 1minute; 94° C. for 30 seconds; 49° C. for 30 seconds; 72° C. for 1minute; repeating the 94° C., 49° C., and 72° C. incubations 30 times;72° C. for 10 minutes and then stopping the reactions. A 15 μl aliquotof each reaction mixture then was electrophoresed through a 1.2% lowmelting point agarose gel in TAE buffer, and then stained with ethidiumbromide. Fragments containing the amplified left and right flanking DNAmolecules were excised from the gel and purified using a QIAQUICK™ gelextraction kit (Qiagen, Inc.) Other art-known methods for amplifying andisolating DNA can be substituted. The flanking left and right DNAfragments were eluted into 30 μl TE buffer at pH 8.0.

[0077] The amplified erm gene and left and right flanking DNA moleculeswere then fused together to produce the fusion product, as shown in FIG.8. The fusion PCR reaction was carried out in a volume of 50 μlcontaining: 2 μl of each of the left and right flanking DNA moleculesand the e=n gene PCR product; 5 μl of lox buffer; 2.5 μl of primer 1 (10pmol/μl); 2.5 μl of primer 4 (10 pmol/μl), 1.2 μl DNTP mix (10 mM each)32 μl H₂O, and 0.7 μl Taq polymerase. The PCR reaction was carried outusing the following cycling program: 95° C. for 2 minutes; 72° C. for 1minute; 94° C. for 30 seconds, 48° C. for 30 seconds; 72° C. for 3minutes; repeat the 94° C., 48° C., and 72° C. incubations 25 times; 72°C. for 10 minutes. After the reaction was stopped, a 12 μl aliquot ofthe reaction mixture was electrophoresed through an agarose gel toconfirm the presence of a final product of approximately 2 kb.

[0078] A 5 μl aliquot of the fusion product was used to transform S.pneumoniae grown on a medium containing erythromycin in accordance withstandard techniques. As shown in FIG. 9, the fusion product and the S.pneumoniae genome undergo a homologous recombination event so that theerm gene replaces the chromosomal copy of the gene of interest, therebycreating a gene knockout. Disruption of an essential gene results in nogrowth on a medium containing erythromycin. Using this gene knockoutmethod, the yphC and yqjK genes were identified as being essential forsurvival. The portion of the yphC open reading frame that was sequencedprior to carrying out targeted deletion is depicted in FIG. 1. Thefull-length yphC sequence (depicted in FIGS. 2A-2B) was compiled bysearching the TIGR sequence database for a clone from S. pneumoniaehaving a sequence overlapping the sequence depicted in FIG. 1 andcombining the 3′ end of the gene from the database with the 5′ end ofthe gene depicted in FIG. 1. The sequence contained in the clone fromthe database was of unknown function.

[0079] Identification of Orthologs of Essential Genes

[0080] Having shown that the yphC and yqjK genes are essential forsurvival of Streptococcus, orthologs of these genes, when identified inother organisms, for example B. subtilis or E. coli, can be tested todetermine whether they are essential for survival of those organisms aswell. To this end, the coding sequences of yphC and yqjK were used tosearch the GenBank database of nucleotide sequences, and orthologs ofeach sequence were identified in B. subtilis and E. coli. Sequencecomparisons were performed using the Basic Local Alignment Search Tool(BLAST) (Altschul et al., J. Mol. Biol. 215:403-410, 1990). The percentsequence identity shared by the essential polypeptides and theirorthologs was determined using the GAP program from the GeneticsComputer Group (GCG) Wisconsin Sequence Analysis Package (WisconsinPackage Version 9.1; Madison, Wisc.). The default parameters for gapweight (12) and length weight (4) were used.

[0081] Typically, essential polypeptides and their orthologs share atleast 25% (e.g., at least 30% or 40%) sequence identity. Typically, theDNA sequences encoding essential polypeptides and their homologs ororthologs share at least 20% (e.g., at least 30%, 35%, 40% or 45%)sequence identity. Bioinformatics analysis of the yphC and yqjK genesshowed that these genes are widely conserved among bacteria.

[0082] To determine whether the identified orthologs of yphC and yqjKare essential for survival of other bacteria, each of the orthologousgenes was separately deleted from the genome of the host organism, asdescribed in detail below. The observation that the B. subtilis and E.coli orthologs of yphC (B-yphC and yfgK, respectively) also areessential for survival of B. subtilis and E. coli suggests that the yphCgene is essential in all bacteria where it is present. Therefore, anantibacterial agent targeted to this gene or its gene product isexpected to have a broad spectrum of antibacterial activity. Theobservation that the B. subtilis ortholog of yqjK (B-yqjK), but not theE. coli ortholog (elaC), is essential for survival suggests that thisgene is essential in all gram-positive bacteria in which it is present,and not essential in gram negative bacteria. Therefore, an antibacterialagent targeted to the yqjK gene or its gene product is expected to haveantibacterial activity against all gram-positive bacteria.

[0083] Deletion and Determination of Essentiality of the yphC and yqjKGenes in Bacillus Subtilis

[0084] The following examples illustrate that the B. subtilis orthologsof yphC and yqjK (i.e., B-yphC and B-yqjK) are essential for survival ofB. subtilis. Two strategies were used to produce knockout mutations ofthe B-yphC and B-yqjK genes in B. subtilis, and a determination of theessential phenotype of the B-yphC and B-yqjK genes was made, asdescribed below. The first strategy (illustrated in FIG. 10) was similarto the targeted deletion strategy used to knock out genes inStreptococcus, as described above. The significant differences were asfollows. (A.) In PCR, a chloramphenicol resistant gene (CmR) of B.subtilis, from plasmid pDG283, was used in lieu of an erythromycinresistance gene. Alternatively, any B. subtilis marker can be used.

[0085] (B.) The primers used to amplify the CmR gene and primers B andC, which immediately flank the yphC ORF, contain a stretch of 27nucleotides termed “universal overlapping sequences.” These universaloverlapping sequences can be used efficiently in PCR amplifications, andfacilitate the use of various insertion sequences in fusion PCRreactions. Resistance markers, promoters, regulatory elements, or anynucleic acid sequence can be amplified with such overlapping sequencesand be used with the same set of gene deletion primers (primers A, B, C,and D). The sequence for the 5′ overlapping region is 5′CACAGGAAACAGCTATGACCATGATTA3′ (SEQ ID NO:25) and the sequence for the 3′ overlappingregion is 5′ GAAATAAATGCATCTGTATTTGAATG3′ (SEQ ID NO:26).

[0086] (C.) The left and right flanking DNA molecules produced by PCRshould be at least 900 (e.g., 1000) nucleotides in length to optimizerecombination into the B. subtilis chromosome.

[0087] (D.) To produce the fusion product, two simultaneous PCRreactions were used. One reaction used an annealing temperature of 5°C., and the other used a temperature of 65° C. Longer extension timeswere used (30-60 more seconds), and a Long high fidelity polymerase wasalso used according to the manufacturer's instructions(Boehringer-Mannheim).

[0088] (E.) Competent cells of the wild-type strain, PY79, were usedaccording to standard B. subtilis protocols (Molecular BiologicalMethods for Bacillus, 1990, Harwood and Cutting, Eds. Wiley & Sons, Ltd.England).

[0089] (F.) The sequence of the primers shown in FIG. 10 was as follows:primer Ra, (5′CACAGGAAACAGCTATGACCA TGATTAAACTAAAGCACCCATTAGTTCA3′ (SEQID NO:27)) which hybridized to a sequence upstream of the CmR promoter;primer Rb (5′CATTCAAATACAGATGCATTTTATTTCCTCATATT ATAAAAGCCAGTCATT3′ (SEQID NO:28)), which hybridized to a sequence located adjacent to thetranscription terminator; primer A-YPHC (5′ GCCATTGCGTTTGAAAG3′ (SEQ IDNO:29)); primer A-YQJK (5′ TGCTTCGCCGATTTCTT3′ (SEQ ID NO:30); primerB-YPHC (5′ TAATCATGGTCATAGCTGTTTCCTGTGTATGAAAAGAAACCCTTCAGAG3′ (SEQ IDNO:31)), which is located adjacent to the yphC start codon; primerB-YQJK (5′TAATCATGGTCATAGCTGTTTCCTGTGCATACCG AACGCCTTTCTT3′ (SEQ IDNO:32)), which is located adjacent to the yqjK start codon; primerC-YPHC (5′GAAATAAATGCA TCTGTATTTGAATGTTTTAGAAAACCGAATCAGAGA3′ (SEQ IDNO:33)), which is located adjacent to the yphC stop codon; primer C-YQJK(5′GAAATAAATGCATCTGTATTTGAATGAATAGCGTGGCGGCATA3′ (SEQ ID NO:34)),located adjacent to the yqjK stop codon; primer D-YPHC(5′ATTCAGATCGAATACTCCTG3′ (SEQ ID NO:35)); and primer D-YQJK (5′AAAGCGGGCAAAGCAGA3′ (SEQ ID NO:36)).

[0090] Competent cells that were transformed with the fused left andright flanking DNA molecules were incubated for 18 hours at 37° C. on aselective medium (LB, 5 μg/ml Cm) to determine whether the gene inquestion was essential (as characterized by lack of colony growth) ornon-essential (as characterized by the appearance of dozens to hundredsof colonies). When these deletion experiments were performed with theyphC and yqjK genes, separately, no colonies were detected on theselective medium, indicating that each of these genes is essential forsurvival of B. subtilis.

[0091] Several control experiments were performed to ensure that theobserved lack of cell growth was due to the essential nature of theB-yphC and B-yqjK genes. Simultaneous PCR reactions and transformationswith genes that were known not to be essential produced hundreds ofcolonies in similar experiments, indicating that experimental conditionswere adequate to ensure that the lack of colony growth was indicative ofan essential gene. The CmR gene insertion was also shown to havenon-polar effects on downstream genes and allowed efficient expressionof downstream genes.

[0092] The second strategy used to obtain deletion mutants of the yphCand yqjK genes of B. subtilis involved the construction of conditionalnull mutants that can halt the expression of the chosen gene and allowobservation of the mutant phenotype (FIGS. 11A-11C). In these mutants, awild-type copy of the yphC or yqjK gene was placed under the control ofa B. subtilis Para promoter, which is a tightly regulatable promoterthat can efficiently turn off gene expression. These regulatable geneticconstructs were subsequently inserted into the B. subtilis chromosome.As shown in FIG. 11A, the regulatable genetic constructs contained anamylase gene (amy) sequence and CmR sequence for integration intochromosome at the amy locus. Disruption of the amy gene by doublecrossover is innocuous to the cell, and recombinants are easilydetectable on starch plates because amy⁺ cells produce colonies havingtransparent halos.

[0093] After integration of the regulatable yphC or yqjK genes into thechromosome of wild-type cells, the resulting cells were renderedcompetent and transformed with a fusion PCR fragment containing areplacement resistant marker (FIG. 11B), as described above. In thiscase, the fusion PCR fragment contained a Kanamycin resistance gene fromB. subtilis (Kan) instead of the CmR gene. Selection for this Kan markerduring transformation of the cells containing the ectopically insertedregulated yphC or yqjK genes was performed in the presence of arabinoseto allow expression of the yphC or yqjK genes and trans complementationof the deletion (FIG. 11C).

[0094] The yphC and yqjK mutants obtained in this manner were able togrow on a selective medium (LB Kan) in the presence of 0.2% arabinose,while selections made in wild type cells did not yield any mutants.Colonies containing the regulated genes and their deletions then werepicked and streaked onto a similar medium (LB Kan, 0.2% arabinose), andonto plates containing a selective medium with lower concentrations ofarabinose, no arabinose, or in the presence of 0.2% glucose. After 18hours of incubation to allow for growth, the only plates that containedcolonies were those plates that contained 0.2%, 0.02%, or 0.002%arabinose. Lower concentrations of the inducer or repressing conditions(i.e., the lack of arabinose or presence of glucose) did not allow cellgrowth and formation of colonies. Thus, these experiments indicatedthat: (i) the deletion created by the fusion PCR did not affect putativeessential genes downstream of yphC or yqjK, since expression of only thegene in question was sufficient to obtain efficient complementation, and(ii) expression of the yphC and the yqjK genes is necessary for survivalof the B. subtilis cells.

[0095] The experiments described above confirmed that B-yphC and B-yqjKare essential in B. subtilis. These experiments yielded conditionallethal strains that can be used in a variety of screens and approaches,including underexpression/overexpression assays, transcriptionprofiling, etc. The constructions of knockout mutations of the yphC andyqjK genes can be accomplished using any of various art-known methods.

[0096] Assay of the Essential Nature of yphC and yqjK Orthologs in E.coli

[0097] The discovery that the yphC and yqjK genes are essential inStreptococcus pneumoniae and in B. subtilis suggests that these genesare essential in all Gram-positive bacteria. To further extend theseobservations to Gram-negative bacteria, and therefore to all bacteria,deletion mutants were produced for the E. coli orthologs of yphC andyqjK.

[0098] The general strategy used to produce E. coli gene deletions, asconditional null mutants, is schematically represented in FIG. 12.First, a copy of the wild type gene to be deleted was cloned into arunaway, counter-selectable vector containing the Pbad promoter of E.coli. This E. coli promoter is turned on in the presence of arabinoseand is tightly controlled like its B. subtilis counterpart, describedabove. Cells containing this vector were then used to introduce anin-frame deletion of the chosen gene by replacing the gene with amarkerless small TAG of approximately 30 nucleotides.

[0099] The upstream and downstream regions that flank the chosen genewere amplified by PCR using primers that introduced a 27-30 nucleotideoverlapping TAG. Fusion PCR reactions were carried out with only thesetwo fragments that are joined with the TAG lying in the middle, therebyreplacing the chosen gene.

[0100] This fragment was then cloned into a temperature-sensitive,counterselectable plasmid, pKO-3, and inserted into the chromosome inaccordance with conventional techniques (see, e.g., Church et al., 1997,J. Bacteriol.). The resulting in-frame deletion was complemented byexpression of the wild type gene from the Pbad vector in the presence ofarabinose (FIG. 11C, P turned on. The deletion mutant can suppress geneexpression under conditions lacking arabinose, in the presence ofglucose (P turned off), or in the presence of streptomycin without IPTG,which allows loss of the plasmid (because the origin of replication ofthe complementing Pbad plasmid is under lac-IPTG control).

[0101] As shown in FIG. 12, deletion of the yphC sequence in E. coli andits substitution by a TAG in the presence of a complementing Pbad-yphCortholog plasmid resulted in mutants that grew well on arabinose platesbut which failed to grow on glucose or streptomycin plates. This resultindicates that the yfgK gene (i.e., the E. coli yphC ortholog) isessential for the survival of E. coli. In contrast, similar experimentscarried out with the E. coli yqjK ortholog, elaC, showed significantcell growth under all conditions, indicating that the yqjK gene is notessential in E. coli. Alternatively, it is possible that other E. coligenes that have arylsulfatase/phosphatase activity that have no sequencesimilarity are able to complement for the lack of elaC function.

[0102] The fact that the B. subtilis and E. coli orthologs of the yphCgene are essential for survival indicates that this gene is essential inall bacteria in which it is present. The yqjK gene, which is essentialfor survival of S. pneumoniae and B. subtilis, is thought to beessential in all Gram-positive bacteria, but not in E. coli. Therefore,an antibacterial agent targeted to the yphC gene or its gene product isexpected to have a broad spectrum of antibacterial activity (includingGram-positive and Gram-negative bacteria), while an antibacterial agenttargeted to the yqjK gene or its gene product is expected to haveantibacterial activity against Gram-positive bacteria.

[0103] Identification of Essential Genes and Polypeptides in AdditionalBacterial Strains

[0104] The yphC and yqjK genes and various orthologs, or fragmentsthereof, can be used to detect homologous or orthologous genes in yetother organisms. In particular, these genes can be used to analyzevarious pathogenic and non-pathogenic strains of bacteria. Fragments ofa nucleic acid (DNA or RNA) encoding an essential polypeptide, homologor ortholog (or sequences complementary thereto) can be used as probesin conventional nucleic acid hybridization assays of pathogenicbacteria. For example, nucleic acid probes (which typically are 8-30,usually 15-20, nucleotides in length) can be used to detect essentialgenes or homologs or orthologs thereof in art-known molecular biologymethods, such as Southern blotting, Northern blotting, dot or slotblotting, PCR amplification methods, colony hybridization methods, andthe like. Typically, an oligonucleotide probe based on the nucleic acidsequences described herein, or fragments thereof, is labeled and used toscreen a genomic library constructed from mRNA obtained from a bacterialstrain of interest. A suitable method of labeling involves usingpolynucleotide kinase to add ³²P-labeled ATP to the oligonucleotide usedas the probe. This method is well known in the art, as are several othersuitable methods (e.g., biotinylation and enzyme labeling).

[0105] Hybridization of the oligonucleotide probe to the library, orother nucleic acid sample, typically is performed under moderate tostringent conditions. Nucleic acid duplex or hybrid stability isexpressed as the melting temperature or T_(m) which is the temperatureat which a probe dissociates from a target DNA. This melting temperatureis used to define the required stringency conditions. If sequences areto be identified that are related and substantially identical to theprobe, rather than identical, it is useful to first establish the lowesttemperature at which only homologous hybridization occurs with aparticular concentration of salt (e.g., SSC or SSPE). Then, assumingthat 1% mismatching results in a 1° C. decrease in the T_(m), thetemperature of the final wash in the hybridization reaction is reducedaccordingly (for example, if sequences having >95% identity with theprobe are sought, the final wash temperature is decreased by 5° C.). Inpractice, the change in T_(m) can be between 0.50 and 1.5° C. per 1%mismatch.

[0106] Stringent conditions include, for example, hybridizing at 68° C.in 5× SSC/5× Denhardt's solution/1.0% SDS, or in 0.5 M NaHPO₄ (pH 7.2)/1mM EDTA/7% SDS, or in 50% formamide/0.25 M NaHPO₄ (pH 7.2)/0.25 M NaCl/1mM EDTA/7% SDS; and washing in 0.2× SSC/0.1% SDS at room temperature orat 42° C., or in 0.1× SSC/0.1% SDS at 68° C., or in 40 mM NaHPO₄ (pH7.2)/1 mM EDTA/5% SDS at 50° C., or in 40 mM NaHPO₄ (pH 7.2) 1 mMEDTA/1% SDS at 50° C. Moderately stringent conditions include washing in3× SSC at 42° C. The parameters of salt concentration and temperaturecan be varied to achieve the optimal level of identity between the probeand the target nucleic acid. Additional guidance regarding suchconditions is readily available in the art, for example, by Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols inMolecular Biology, (John Wiley & Sons, N.Y.) at Unit 2.10.

[0107] In one approach, libraries constructed from pathogenic ornon-pathogenic bacterial strains can be screened. For example, suchstrains can be screened for expression of essential genes by Northernblot analysis. Upon detection of transcripts of the essential genes orhomologs thereof, libraries can be constructed from RNA isolated fromthe appropriate strain, utilizing standard techniques well known tothose of skill in the art. Alternatively, a total genomic DNA librarycan be screened using an essential gene probe (or a probe directed to ahomolog thereof).

[0108] New gene sequences can be isolated, for example, by performingPCR using two degenerate oligonucleotide primer pools designed on thebasis of nucleotide sequences within the essential genes, or theirhomologs or orthologs, as depicted herein. The template for the reactioncan be DNA obtained from strains known or suspected to express anessential allele or an allele of a homolog or ortholog thereof. The PCRproduct can be subcloned and sequenced to ensure that the amplifiedsequences represent the sequences of a new essential nucleic acidsequence, or a sequence of a homolog thereof.

[0109] Synthesis of the various essential polypeptides or their homologsor orthologs (or an antigenic fragment thereof) for use as antigens, orfor other purposes, can readily be accomplished using any of the variousart-known techniques. For example, an essential polypeptide or homologor ortholog thereof, or an antigenic fragment(s), can be synthesizedchemically in vitro, or enzymatically (e.g., by in vitro transcriptionand translation). Alternatively, the gene can be expressed in, and thepolypeptide purified from, a cell (e.g., a cultured cell) by using anyof the numerous, available gene expression systems. For example, thepolypeptide antigen can be produced in a prokaryotic host (e.g., E. colior B. subtilis) or in eukaryotic cells, such as yeast cells or in insectcells (e.g., by using a baculovirus-based expression vector).

[0110] Proteins and polypeptides can also be produced in plant cells, ifdesired. For plant cells, viral expression vectors (e.g., cauliflowermosaic virus and tobacco mosaic virus) and plasmid expression vectors(e.g., Ti plasmid) are suitable. Such cells are available from a widerange of sources (e.g., the American Type Culture Collection, Rockland,Md.; also, see, e.g., Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1994). The optimal methods oftransformation or transfection and the choice of expression vehicle willdepend on the host system selected. Transformation and transfectionmethods are described, e.g., in Ausubel et al., supra; expressionvehicles may be chosen from those provided, e.g., in Cloning Vectors: ALaboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987). The hostcells harboring the expression vehicle can be cultured in conventionalnutrient media, adapted as needed for activation of a chosen gene,repression of a chosen gene, selection of transformants, oramplification of a chosen gene.

[0111] If desired, the yphC or yqjK polypeptides or their homologs ororthologs can be produced as fusion proteins. For example, theexpression vector pUR278 (Ruther et al., EMBO J., 2:1791, 1983) can beused to create lacZ fusion proteins. The art-known pGEX vectors can beused to express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan be easily purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

[0112] In an exemplary expression system, a baculovirus such asAutographa californica nuclear polyhedrosis virus (AcNPV), which growsin Spodoptera frugiperda cells, can be used as a vector to expressforeign genes. A coding sequence encoding an essential polypeptide orhomolog or ortholog thereof can be cloned into a non-essential region(for example the polyhedrin gene) of the viral genome and placed undercontrol of a promoter, e.g., the polyhedrin promoter or an exogenouspromoter. Successful insertion of a gene encoding an essentialpolypeptide or homolog can result in inactivation of the polyhedrin geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat encoded by the polyhedrin gene). Theserecombinant viruses are then typically used to infect insect cells(e.g., Spodoptera frugiperda cells) in which the inserted gene isexpressed (see, e.g., Smith et al., J. Virol., 46:584, 1983; Smith, U.S.Pat. No. 4,215,051). If desired, mammalian cells can be used in lieu ofinsect cells, provided that the virus is engineered such that the geneencoding the desired polypeptide is placed under the control of apromoter that is active in mammalian cells.

[0113] In mammalian host cells, a number of viral-based expressionsystems can be utilized. When an adenovirus is used as an expressionvector, the nucleic acid sequence encoding the essential polypeptide orhomolog can be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene can then be inserted into the adenovirus genome by invitro or in vivo recombination. Insertion into a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing an essential gene productin infected hosts (see, e.g., Logan, Proc. Natl. Acad. Sci. USA,81:3655, 1984).

[0114] Specific initiation signals may be required for efficienttranslation of inserted nucleic acid sequences. These signals includethe ATG initiation codon and adjacent sequences. In general, exogenoustranslational control signals, including, perhaps, the ATG initiationcodon, should be provided. Furthermore, the initiation codon must be inphase with the reading frame of the desired coding sequence to ensuretranslation of the entire sequence. These exogenous translationalcontrol signals and initiation codons can be of a variety of origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of appropriate transcription enhancer elements, ortranscription terminators (Bittner et al., Methods in Enzymol., 153:516,1987).

[0115] The essential polypeptides and their homologs and orthologs canbe expressed individually or as fusions with a heterologous polypeptide,such as a signal sequence or other polypeptide having a specificcleavage site at the N-and/or C-terminus of the protein or polypeptide.The heterologous signal sequence selected should be one that isrecognized and processed, i.e., cleaved by a signal peptidase, by thehost cell in which the fusion protein is expressed.

[0116] A host cell can be chosen that modulates the expression of theinserted sequences, or modifies and processes the gene product in aspecific, desired fashion. Such modifications and processing (e.g.,cleavage) of protein products may facilitate optimal functioning of theprotein. Various host cells have characteristic and specific mechanismsfor post-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems familiar to those ofskill in the art of molecular biology can be chosen to ensure thecorrect modification and processing of the foreign protein expressed. Tothis end, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, and phosphorylation of thegene product can be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroidplexus cell lines.

[0117] If desired, the essential polypeptide or homolog or orthologthereof can be produced by a stably-transfected mammalian cell line. Anumber of vectors suitable for stable transection of mammalian cells areavailable to the public, see, e.g., Pouwels et al. (supra); methods forconstructing such cell lines are also publicly known, e.g., in Ausubelet al. (supra). In one example, DNA encoding the protein is cloned intoan expression vector that includes the dihydrofolate reductase (DHFR)gene. Integration of the plasmid and, therefore, the essentialpolypeptide-encoding gene into the host cell chromosome is selected forby including 0.01-300 μM methotrexate in the cell culture medium (asdescribed in Ausubel et al., supra). This dominant selection can beaccomplished in most cell types.

[0118] Recombinant protein expression can be increased by DHFR-mediatedamplification of the transfected gene. Methods for selecting cell linesbearing gene amplifications are described in Ausubel et al. (supra);such methods generally involve extended culture in medium containinggradually increasing levels of methotrexate. DHFR-containing expressionvectors commonly used for this purpose include pCVSEII-DHFR andpAdD26SV(A) (described in Ausubel et al., supra).

[0119] A number of other selection systems can be used, including butnot limited to, herpes simplex virus thymidine kinase genes,hypoxanthine-guanine phosphoribosyl-transferase genes, and adeninephosphoribosyltransferase genes, which can be employed in tk, hgprt, oraprt cells, respectively. In addition, gpt, which confers resistance tomycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA, 78:2072,1981); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., J. Mol. Biol., 150:1, 1981); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene, 30:147, 1981),can be used.

[0120] Alternatively, any fusion protein can be readily purified byutilizing an antibody or other molecule that specifically binds thefusion protein being expressed. For example, a system described inJanknecht et al., Proc. Natl. Acad. Sci. USA, 88:8972 (1981), allows forthe ready purification of non-denatured fusion proteins expressed inhuman cell lines. In this system, the gene of interest is subcloned intoa vaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns,and histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

[0121] Alternatively, yphC or yqjK, or a homolog or ortholog thereof, ora portion thereof, can be fused to an immunoglobulin Fc domain. Such afusion protein can be readily purified using a protein A column, forexample. Moreover, such fusion proteins permit the production of achimeric form of an essential polypeptide or homolog or ortholog havingincreased stability in vivo.

[0122] Once the recombinant essential polypeptide (or homolog) isexpressed, it can be isolated (i.e., purified). Secreted forms of thepolypeptides can be isolated from cell culture media, while non-secretedforms must be isolated from the host cells. Polypeptides can be isolatedby affinity chromatography. For example, an anti-yphC antibody (e.g.,produced as described herein) can be attached to a column and used toisolate the protein. Lysis and fractionation of cells harboring theprotein prior to affinity chromatography can be performed by standardmethods (see, e.g., Ausubel et al., supra). Alternatively, a fusionprotein can be constructed and used to isolate an essential polypeptide(e.g., a yphC-maltose binding fusion protein, a yphC-β-galactosidasefusion protein, or a yphC-trpE fusion protein; see, e.g., Ausubel etal., supra; New England Biolabs Catalog, Beverly, Mass.). Therecombinant protein can, if desired, be further purified, e.g., by highperformance liquid chromatography using standard techniques (see, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology,eds., Work and Burdon, Elsevier, 1980).

[0123] Given the amino acid sequences described herein, polypeptidesuseful in practicing the invention, particularly fragments of essentialpolypeptides, can be produced by standard chemical synthesis (e.g., bythe methods described in Solid Phase Peptide Synthesis, 2nd ed., ThePierce Chemical Co., Rockford, Ill., 1984) and used as antigens, forexample.

[0124] Antibodies

[0125] The yphC and yqjK polypeptides (or antigenic fragments or analogsof such polypeptides) can be used to raise antibodies useful in theinvention, and such polypeptides can be produced by recombinant orpeptide synthetic techniques (see, e.g., Solid Phase Peptide Synthesis,supra; Ausubel et al., supra). Likewise, antibodies can be raisedagainst homologs or orthologs of yphC and yqjK (or antigenic fragmentsand analogs of such homologs and orthologs). In general, thepolypeptides can be coupled to a carrier protein, such as KLH, asdescribed in Ausubel et al., supra, mixed with an adjuvant, and injectedinto a host mammal. A “carrier” is a substance that confers stabilityon, and/or aids or enhances the transport or immunogenicity of, anassociated molecule. Antibodies can be purified, for example, byaffinity chromatography methods in which the polypeptide antigen isimmobilized on a resin.

[0126] In particular, various host animals can be immunized by injectionof a polypeptide of interest. Examples of suitable host animals includerabbits, mice, guinea pigs, and rats. Various adjuvants can be used toincrease the immunological response, depending on the host species,including but not limited to Freund's (complete and incompleteadjuvant), adjuvant mineral gels such as aluminum hydroxide, surfaceactive substances such as lysolecithin, pluronic polyols, polyanions,peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

[0127] Antibodies useful in the invention include monoclonal antibodies,polyclonal antibodies, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, and molecules producedusing a Fab expression library.

[0128] Monoclonal antibodies (mAbs), which are homogeneous populationsof antibodies to a particular antigen, can be prepared using yphC, yqjK,or homologs or orthologs thereof and-standard-hybridoma-technology (see,e.g., Kohler et al., Nature, 256:495, 1975; Kohler et al., Eur. J.Immunol., 6:511, 1976; Kohler et al., Eur. J. Immunol., 6:292, 1976;Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas,Elsevier, N.Y., 1981; Ausubel et al., supra).

[0129] In particular, monoclonal antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture, such as those described in Kohler etal., Nature, 256:495, 1975, and U.S. Pat. No. 4,376,110; the humanB-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72, 1983;Cole et al., Proc. Natl. Acad. Sci. USA, 80:2026, 1983); and theEBV-hybridoma technique (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96, 1983). Such antibodies can be ofany immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and anysubclass thereof. The hybridomas producing the mAbs of this inventioncan be cultivated in vitro or in vivo.

[0130] Once produced, polyclonal or monoclonal antibodies are tested forspecific recognition of an essential polypeptide or homolog or orthologthereof in an immunoassay, such as a Western blot or immunoprecipitationanalysis using standard techniques, e.g., as described in Ausubel etal., supra. Antibodies that specifically bind to the essentialpolypeptides, or conservative variants and homologs and orthologsthereof, are useful in the invention. For example, such antibodies canbe used in an immunoassay to detect an essential polypeptide inpathogenic or non-pathogenic strains of bacteria.

[0131] Preferably, antibodies of the invention are produced usingfragments of the essential polypeptides that appear likely to beantigenic, by criteria such as high frequency of charged residues. Inone specific-example, such fragments are generated by standardtechniques of PCR, and are then cloned into the pGEX expression vector(Ausubel et al., supra). Fusion proteins are expressed in E. coli andpurified using a glutathione agarose affinity matrix as described inAusubel, et al., supra.

[0132] If desired, several (e.g., two or three) fusions can be generatedfor each protein, and each fusion can be injected into at least tworabbits. Antisera can be raised by injections in a series, typicallyincluding at least three booster injections. Typically, the antisera ischecked for its ability to immunoprecipitate a recombinant essentialpolypeptide or homolog, or unrelated control proteins, such asglucocorticoid receptor, chloramphenicol acetyltransferase, orluciferase.

[0133] Techniques developed for the production of “chimeric antibodies”(Morrison et al., Proc. Natl. Acad. Sci., 81:6851, 1984; Neuberger etal., Nature, 312:604, 1984; Takeda et al., Nature, 314:452, 1984) can beused to splice the genes from a mouse antibody molecule of appropriateantigen specificity together with genes from a human antibody moleculeof appropriate biological activity. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a murine mAb and a humanimmunoglobulin constant region.

[0134] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. Nos. 4,946,778; 4,946,778; and 4,704,692)can be adapted to produce single chain antibodies against an essentialpolypeptide or homolog or ortholog thereof. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

[0135] Antibody fragments that recognize and bind to specific epitopescan be generated by known techniques. For example, such fragments caninclude but are not limited to F(ab′)₂ fragments, which can be producedby pepsin digestion of the antibody molecule, and Fab fragments, whichcan be generated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science, 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0136] Polyclonal and monoclonal antibodies that specifically bind toessential polypeptides, homologs, or orthologs can be used, for example,to detect expression of an essential gene, homolog, or ortholog inanother bacteria. For example, an essential polypeptide can be readilydetected in conventional immunoassays of bacteria cells or extracts.Examples of suitable assays include, without limitation, Westernblotting, ELISAs, radioimmune assays, and the like.

[0137] Assay for Antibacterial Agents

[0138] The invention provides methods for identifying antibacterialagents. Without being bound by any particular theory as to thebiological mechanism involved, the new antibacterial agents are thoughtto inhibit specifically (1) the function of the yphC or yqjKpolypeptide(s), or homologs or orthologs thereof, or (2B) expression ofthe yphC or yqjK genes, or homologs or orthologs thereof.

[0139] Alignment of the yphC protein sequence with similar sequencesfrom the GenBank database suggests that the yphC protein has GTPaseactivity. Similarly, the alignment of the yqjK protein with sequencesfrom GenBank suggests that the yqjK protein has arylsulfatase activity.In experiments designed to test whether the yphC and yqjK proteins havethe proposed biochemical activities, the yphC protein was shown to haveGTPase activity, and the yqjK protein was shown to have arylsulfataseactivity.

[0140] The enzymatic activity of each protein suggests novel features ofeach enzyme. For example, the yphC protein contains two GTP-bindingsites, but only one of the sites appears to be active. The yqjK proteinhas phosphatase activity in addition to arylsulfatase activity, and theprotein carries out an activation step that is catalyzed by manganeseions. In the genetic experiments described herein, the observedbiochemical activities of yphC and yqjK were shown to be linked to theessential nature of the proteins. Mutants of the GTP-binding sites ofthe yphC gene result in proteins that lack GTPase activity and that areunable to complement null yphC mutants. Similarly, yqjK mutants thatlack arylsulfatase activity are unable to complement null yqjK mutants.Additionally, mutants that lacked arylsulfatase activity also lackedphosphatase activity and vice versa. These experiments indicate thatinhibition of the enzymatic activities of yphC or yqjK in cell culturesimpairs the viability of the cells and results in cell death. Thus,these biochemical activities can be used in vivo or in vitro, alone orin combination with any art-known methods to detect these activities, toidentify antibacterial agents.

[0141] In various suitable methods, screening for antibacterial agentsis accomplished by identifying those compounds (e.g., small organicmolecules) that inhibit the activity of an essential polypeptide or theexpression of an essential gene. Screening for antibacterial agents canbe accomplished by (i) identifying those compounds that interact with orbind to an essential polypeptide and (ii) further testing such compoundsfor their ability to inhibit bacterial growth in vitro or in vivo.

[0142] Examples of suitable screening methods are set forth in U.S. Pat.Nos. 5,679,582 and 5,585,277, which are incorporated herein byreference. Briefly, in these methods, a target protein is incubated inthe presence of a test compound (i.e., test ligand) to produce a “testcombination,” and the target protein is incubated in the absence of atest compound to produce a “control combination.” The test and controlcombinations are then treated to cause a detectable fraction of thetarget protein to exist in a partially or totally unfolded state. Theextent to which the target protein occurs in a folded state, an unfoldedstate, or both, in the test and control combinations is then determined.When the target protein is present in the folded state to a greater orlesser extent in the test combination than in the control combination,the test compound is a compound that binds the target protein.

[0143] In an alternative method, binding of a test compound to a targetprotein is detected using capillary electrophoresis. Briefly, testcompounds (e.g., small molecules) that bind to the target protein causea change in the electrophoretic mobility of the target protein duringcapillary electrophoresis. Suitable capillary electrophoresis methodsare known in the art (see, e.g., Freitag, J. Chromatography B,Biomedical Sciences & Applications: 722(1-2B):279-301, Feb. 5, 1999; Chuand Cheng, Cellular & Molecular Life Sciences: 54(7):663-83, July 1998;Thormann et al., Forensic Science International: 92(2-3): 157-83, Apr.5, 1998; Rippel et al., Electrophoresis: -18(12-13)-: 21-75-83, Nova1997;Hage and Tweed J. Chromatography. B, Biomedical Sciences &Applications: 699(1-2B):499-525, Oct. 10, 1997; Mitchelson et al.,Trends In Biotechnology: 15(11):448-58, Nov. 1997; Jenkins and Guerin J.Chromatography B. Biomedical Applications: 682(1):23-34, Jun. 28, 1996;and Chen and Gallo, Electrophoresis: 19(16-17):2861-9, Nov. 1998.

[0144] Inhibitors of yphC can also be identified in the followingbiochemical assay for detection of GTPase inhibitors. This assay uses acalorimetric detection system for the detection of nanomolar amounts ofinorganic phosphate. The assay can be carried out in a clear bottom96-well microplate (e.g., Corning-COSTAR Catalog #9710). A 20 μl aliquotof each test compound in 10% DMSO is placed into each well of the plate,except those wells that are used as controls. A 20 μl aliquot of 420 μMGTP then is added to each well of the plate, except those wells that areused for control reactions. 20 μl of IC₅₀ controls, containing 225 μMGDP, is dispensed into two of the control wells; 20 μl of IC₁₀₀ (2.25 mlof 0.5M EDTA in 12.75 ml of 420 μM GTP) is dispensed into two othercontrol wells; and 20 μl of no inhibition controls containing 420 μM GTPis dispensed into four other control wells. 20 μl of 2BX-buffer (100 mMTris HCL, 500 mM KCl, 10 mM MGCl₂, 0.2 mg/ml Acetylated-BSA, H₂O) plusyphC enzyme solution (to provide 1 μg/well) then is dispensed into eachwell, and the plate is incubated at room temperature for 3.5 hours. Tostop the enzyme reaction, 150 μl of 0.045% Malachite Green/35 mM EDTAsolution is added to each well. After 25 minutes, 50 μl of 15% citrateis added to each well to prevent further color development. The samplesthen are mixed vigorously (e.g., with a TOMTEC-Quadra-96, Model 320)until a homogenous solution results. The plates subsequently are readusing a plate reader (e.g., a Wallac-Victor 2 plate reader) set at awavelength of 650 nm. Generally, the plates should be read within 24hours of adding the Malachite Green and citrate.

[0145] The percent inhibition for each sample well can be calculated asfollows. The average of the two wells that contained IC₁₀₀ controls canbe used as the background counts. The percent inhibition can calculatedaccording the following formula:

% inhibition=[1−(sample counts−background counts)/(averagecounts−background counts)]×100.

[0146] Test compounds that produce greater than 40% inhibition may beretested with a dose response at a higher concentration, if desired.

[0147] Other methods for identifying antibacterial agents includevarious cell-based methods for identifying polypeptides that bind yphC,yqjK, or homologs or orthologs thereof, such as the conventionaltwo-hybrid assays of protein/protein interactions (see e.g., Chien etal., Proc. Natl. Acad. Sci. USA, 88:9578, 1991; Fields et al., U.S. Pat.No. 5,283,173; Fields and Song, Nature, 340:245, 1989; Le Douarin etal., Nucleic Acids Research, 23:876, 1995; Vidal et al., Proc. Natl.Acad. Sci. USA, 93:10315-10320, 1996; and White, Proc. Natl. Acad. Sci.USA, 93:10001-10003, 1996). Generally, the two-hybrid methods involvereconstitution of two separable domains of a transcription factor in acell. One fusion protein contains the essential polypeptide (or homologor ortholog thereof) fused to either a transactivator domain or DNAbinding domain of a transcription factor (e.g., of Gal4). The otherfusion protein contains a test polypeptide fused to either the DNAbinding domain or a transactivator domain of a transcription factor.Once brought together in a single cell (e.g., a yeast cell or mammaliancell), one of the fusion proteins contains the transactivator domain andthe other fusion protein contains the DNA binding domain. Therefore,binding of the essential polypeptide to the test polypeptide (i.e.,candidate antibacterial agent) reconstitutes the transcription factor.Reconstitution of the transcription factor can be detected by detectingexpression of a gene (i.e., a reporter gene) that is operably linked toa DNA sequence that is bound by the DNA binding domain of thetranscription factor. Kits for practicing various two-hybrid methods arecommercially available (e.g., from Clontech; Palo Alto, Calif.).

[0148] In another exemplary assay, but not the only assay, a promoterthat responds to depletion of the essential polypeptide by upregulationor downregulation is linked to a reporter gene (e.g., β-galactosidase,gus, or GFP), as described above. A bacterial strain containing thisreporter gene construct is then exposed to test compounds. Compoundsthat inhibit the essential polypeptide (or other polypeptides in theessential pathway in which the essential polypeptide participates) willcause a functional depletion of the essential polypeptide and thereforelead to an upregulation or downregulation of expression the reportergene. Because the polypeptides described herein are essential for thesurvival of bacteria, compounds that inhibit the essential polypeptidesin such an assay are expected to be antibacterial agents and can befurther tested, if desired, in conventional susceptibility assays.

[0149] The methods described above can be used for high throughputscreening of numerous test compounds to identify candidate antibacterial(or anti-bacterial) agents. Having identified a test compound as acandidate antibacterial agent, the candidate antibacterial agent can befurther tested for inhibition of bacterial growth in vitro or in vivo(e.g., using an animal, e.g., rodent, model system) if desired. Usingother, art-known variations of such methods, one can test the ability ofa nucleic acid (e.g., DNA or RNA) used as the test compound to bindyphC, yqjK, or a homolog or ortholog thereof.

[0150] In vitro, further testing can be accomplished by means known tothose in the art such as an enzyme inhibition assay or a whole-cellbacterial growth inhibition assay. For example, an agar dilution assayidentifies a substance that inhibits bacterial growth. Microtiter platesare prepared with serial dilutions of the test compound, adding to thepreparation a given amount of growth substrate, and providing apreparation of bacteria. Inhibition of bacterial growth is determined,for example, by observing changes in optical densities of the bacterialcultures.

[0151] Inhibition of bacterial growth is demonstrated, for example, bycomparing (in the presence and absence of a test compound) the rate ofgrowth or the absolute growth of bacterial cells. Inhibition includes areduction of one of the above measurements by at least 20%. Particularlypotent test compounds may further reduce the growth rate (e.g., by atleast 25%, 30%, 40%, 50%, 75%, 80%, or 90%).

[0152] Rodent (e.g., murine) and rabbit animal models of bacterialinfections are known to those of skill in the art, and such animal modelsystems are accepted for screening antibacterial agents as an indicationof their therapeutic efficacy in human patients. In a typical in vivoassay, an animal is infected with a pathogenic strain of bacteria, e.g.,by inhalation of bacteria such as Streptococcus pneumoniae, andconventional methods and criteria are used to diagnose the mammal asbeing afflicted with a bacterial infection. The candidate antibacterialagent then is administered to the mammal at a dosage of 1-100 mg/kg ofbody weight, and the mammal is monitored for signs of amelioration ofdisease. Alternatively, the test compound can be administered to themammal prior to infecting the mammal with the bacteria, and the abilityof the treated mammal to resist infection is measured. Of course, theresults obtained in the presence of the test compound should be comparedwith results in control animals, which are not treated with the testcompound. Administration of candidate antibacterial agents to the mammalcan be carried out as described below, for example.

[0153] Pharmaceutical Formulations

[0154] Treatment includes administering a pharmaceutically effectiveamount of a composition containing an antibacterial agent to a subjectin need of such treatment, thereby inhibiting bacterial growth in thesubject. Such a composition typically contains from about 0.1 to 90% byweight (such as 1 to 20% or 1 to 10%) of an antibacterial agent of theinvention in a pharmaceutically acceptable carrier.

[0155] Solid formulations of the compositions for oral administrationmay contain suitable carriers or excipients, such as corn starch,gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin,mannitol, dicalcium phosphate, calcium carbonate, sodium chloride, oralginic acid. Disintegrators that can be used include, withoutlimitation, micro-crystalline cellulose, corn starch, sodium starchglycolate and alginic acid. Tablet binders that may be used includeacacia, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose,starch, and ethylcellulose. Lubricants that may be used includemagnesium stearates, stearic acid, silicone fluid, talc, waxes, oils,and colloidal silica.

[0156] Liquid formulations of the compositions for oral administrationprepared in water or other aqueous vehicles may contain varioussuspending agents such as methylcellulose, alginates, tragacanth,pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, and polyvinylalcohol. The liquid formulations may also include solutions, emulsions,syrups and elixirs containing, together with the active compound(s),wetting agents, sweeteners, and coloring and flavoring agents. Variousliquid and powder formulations can be prepared by conventional methodsfor inhalation into the lungs of the mammal to be treated.

[0157] Injectable formulations of the compositions may contain variouscarriers such as vegetable oils, dimethylacetamide, dimethylformamide,ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, liquid polyethylene glycol, and the like).For intravenous injections, water soluble versions of the compounds maybe administered by the drip method, whereby a pharmaceutical formulationcontaining the antibacterial agent and a physiologically acceptableexcipient is infused. Physiologically acceptable excipients may include,for example, 5% dextrose, 0.9% saline, Ringer's solution or othersuitable excipients. Intramuscular preparations, a sterile formulationof a suitable soluble salt form of the compounds can be dissolved andadministered in a pharmaceutical excipient such as Water for Injection,0.9% saline, or 5% glucose solution. A suitable insoluble form of thecompound may be prepared and administered as a suspension in an aqueousbase or a pharmaceutically acceptable oil base, such as an ester of along chain fatty acid, (e.g., ethyl oleate).

[0158] A topical semi-solid ointment formulation typically contains aconcentration of the active ingredient from about 1 to 20%, e.g., 5 to10% in a carrier such as a pharmaceutical cream base. Variousformulations for topical use include drops, tinctures, lotions, creams,solutions, and ointments containing the active ingredient and varioussupports and vehicles.

[0159] The optimal percentage of the antibacterial agent in eachpharmaceutical formulation varies according to the formulation itselfand the therapeutic effect desired in the specific pathologies andcorrelated therapeutic regimens. Appropriate dosages of theantibacterial agents can be readily determined by those of ordinaryskill in the art of medicine by monitoring the mammal for signs ofdisease amelioration or inhibition, and increasing or decreasing thedosage and/or frequency of treatment as desired. The optimal amount ofthe antibacterial compound used for treatment of conditions caused by orcontributed to by bacterial infection may depend upon the manner ofadministration, the age and the body weight of the subject, and thecondition of the subject to be treated. Generally, the antibacterialcompound is administered at a dosage of 1 to 100 mg/kg of body weight,and typically at a dosage of 1 to 10 mg/kg of body weight.

[0160] Other Embodiments

[0161] The invention also features fragments, variants, analogs, andderivatives of the polypeptides described above that retain one or moreof the biological activities of the yphC and yqjK polypeptides, e.g.,GTPase or sulfatase activities. Included within the invention arenaturally-occurring and non-naturally-occurring variants. Compared withthe naturally-occurring essential gene sequences depicted in FIGS. 1 and3, the nucleic acid sequences encoding variants may have a substitution,deletion, or addition of one or more nucleotides. The preferred variantsretain a function of an essential polypeptide, e.g., as determined in acomplementation assay.

[0162] It is to be understood that, while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims. For example, other art-known assays to detectinteractions of test compounds with proteins, or to detect inhibition ofbacterial growth also can be used with the essential genes, geneproducts, and homologs and orthologs thereof.

What is claimed is:
 1. An isolated nucleic acid molecule that is at least 85% identical to SEQ ID NO:1 ; SEQ ID NO:4; or SEQ ID NO:7.
 2. An isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes: a yphC polypeptide comprising the amino acid sequence of SEQ ID NO:2, as depicted in FIG. 1; a yphC polypeptide comprising the amino acid sequence of SEQ ID NO:5, as depicted in FIGS. 2A-2B; or a yqjK polypeptide comprising the amino acid sequence of SEQ ID NO:8, as depicted in FIG.
 3. 3. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: (1) the sequence of SEQ ID NO:1, as depicted in FIG. 1, or degenerate variants thereof; (2) the sequence of SEQ ID NO:1, as depicted in FIG. 1, or degenerate variants thereof, wherein T is replaced by U; (3) nucleic acid sequences complementary to sequences of (1) and (2B); (4) nucleic acid fragments of sequences of (1), (2), and (3) that are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the polypeptide of SEQ ID NO:2; (5) the sequence of SEQ ID NO:4, as depicted in FIGS. 2A-2B, or degenerate variants thereof; (6) the sequence of SEQ ID NO:4, as depicted in FIGS. 2A-2B, or degenerate variants thereof, wherein T is replaced by U; (7) nucleic acids complementary to (5) and (6); (8) the sequence of SEQ ID NO:7, as depicted in FIG. 3, or degenerate variants thereof; (9) the sequence of SEQ ID NO:7, or degenerate variants thereof, wherein T is replaced by U; (10) nucleic acid sequences complementary to sequences of (8) and (9); and (11) nucleic acid fragments of sequences of (8), (9), and (10) that are at least 15 base pairs in length and that hybridize under stringent conditions to genomic DNA encoding the polypeptide of SEQ ID NO:8.
 4. An isolated nucleic acid molecule that is at least 15 base pairs in length and hybridizes under stringent conditions to SEQ ID NO:1 or SEQ ID NO:4.
 5. A vector comprising a nucleic acid molecule of claim
 1. 6. A host cell comprising an exogenously introduced nucleic acid molecule of claim
 1. 7. An isolated polypeptide encoded by a nucleic acid molecule of claim
 1. 8. A method for identifying an antibacterial agent, the method comprising: (a) contacting a yphC or yqjK polypeptide with a test compound; and (b) detecting interaction of the test compound with the yphC or yqjK polypeptide, wherein interaction indicates that the test compound is an antibacterial agent.
 9. A method of claim 8, further comprising: (c) determining whether a test compound that interacts with the polypeptide inhibits growth of bacteria, relative to growth of bacteria cultured in the absence of a test compound that interacts with the polypeptide, wherein inhibition of growth indicates that the test compound is an antibacterial agent.
 10. A method of claim 8, wherein the polypeptide is derived from a non-pathogenic bacterial strain.
 11. A method of claim 8, wherein the polypeptide is derived from a pathogenic bacterial strain.
 12. A method of claim 8, wherein the test compound is selected from the group consisting of polypeptides, ribonucleic acids, small molecules, and deoxyribonucleic acids.
 13. A pharmaceutical formulation comprising an antibacterial agent identified by the method of claim 8, and a pharmaceutically acceptable excipient.
 14. A method for treating a bacterial infection in an organism, the method comprising administering to the organism a therapeutically effective amount of the pharmaceutical formulation of claim
 13. 15. The method of claim 14, wherein the organism is a human.
 16. An antibody that specifically binds to a polypeptide of claim
 7. 17. A method of claim 8, wherein the interaction is detected by detecting a decrease in function of the polypeptide contacted with the test compound; and further comprising: (c) determining whether a test compound that decreases function of a contacted polypeptide inhibits growth of bacteria, relative to growth of bacteria cultured in the absence of a test compound that decreases function of a contacted polypeptide, wherein inhibition of growth indicates that the test compound is an antibacterial agent.
 18. A method for identifying an antibacterial agent, the method comprising: (a) contacting a nucleic acid encoding yphC or yqjK with a test compound; and (b) detecting interaction of the test compound with the nucleic acid, wherein interaction indicates that the test compound is an antibacterial agent.
 19. A method of claim 18, further comprising: (c) determining whether a test compound that interacts with the nucleic acid inhibits growth of bacteria, relative to growth of bacteria cultured in the absence of the test compound that interacts the nucleic acid, wherein inhibition of growth indicates that the test compound is an antibacterial agent.
 20. A method for identifying an antibacterial agent, the method comprising: (a) contacting an ortholog of a yphC or yqjK polypeptide with a test compound; and (b) detecting interaction of the test compound with the ortholog, wherein interaction indicates that the test compound is an antibacterial agent.
 21. A method of claim 20, further comprising: (c) determining whether a test compound that interacts with the ortholog inhibits growth of bacteria, relative to growth of bacteria cultured in the absence of the test compound that interacts with the ortholog, wherein inhibition of growth indicates that the test compound is an antibacterial agent.
 22. A method of claim 21, wherein the ortholog is selected from the group consisting of B-yphC, B-yqjK, yfgK, and elaC.
 23. A method of claim 21, wherein the ortholog is derived from a pathogenic bacterium.
 24. A method for identifying an antibacterial agent, the method comprising: (a) contacting an ortholog of a yphC or yqjK polypeptide with a test compound; (b) detecting a decrease in function of the ortholog contacted by the test compound; and (c) determining whether a test compound that decreases function of a contacted ortholog inhibits growth of bacteria, relative to growth of bacteria cultured in the absence of a test compound that decreases function of a contacted ortholog, wherein inhibition of growth indicates that the test compound is an antibacterial agent.
 25. A method of claim 24, wherein the ortholog is selected from the group consisting of B-yphC, B-yqjK, yfgK, and elaC.
 26. A method for identifying an antibacterial agent, the method comprising: (a) contacting a nucleic acid encoding an ortholog of yphC or yqjK with a test compound; and (b) detecting interaction of the test compound with the nucleic acid, wherein interaction indicates that the test compound is an antibacterial agent.
 27. A method of claim 26, further comprising: (c) determining whether a test compound that interacts with the nucleic acid inhibits growth of bacteria, relative to growth of bacteria cultured in the absence of a test compound that interacts with the nucleic acid, wherein inhibition of growth indicates that the test compound is an antibacterial agent.
 28. A method of claim 27, wherein the ortholog is selected from the group consisting of B-yphC, B-yqjK, yfgK, and elaC.
 29. A method for treating a mammal having a Streptococcus pneumonia infection, the method comprising inhibiting the function of a yphC or yqjK polypeptide in Streptococcus pneumonia infecting the mammal.
 30. An isolated polypeptide encoded by a nucleic acid molecule of claim
 2. 31. An isolated polypeptide encoded by a nucleic acid molecule of claim
 3. 